Genomic instability is postulated to contribute to the aging process. The molecular mechanisms responsible for this increase remain obscure but recent work suggests that DNA damage arising from replication stress and telomere dysfunction contribute to the process. The telomere is a DNA-protein structure located at the termini of linear chromosomes and its maintenance is required for genome stability. Telomere stability is ensured by the presence of sufficient DNA reserves and a growing list of associated proteins. Six proteins, TRF1, TRF2, RAP1, TIN2, TPP1, and POT1 form a core complex that is constitutively present at the telomere and required for telomere maintenance. It was once thought that one function of the Shelterin complex was to exclude replication and repair proteins from the telomere. However, there is now growing appreciation for the importance the Shelterin components play in modulating the activities of many DNA replication and repair proteins to ensure that they contribute to high fidelity telomere replication and maintenance of a stable telomere structure. The current challenge is to elucidate how the Shelterin complex carries out this function at the molecular level. The focus of this proposal is on one such DNA replication and repair protein, human Dna2 (hDna2), which has been implicated in Okazaki fragment processing, DNA repair, and telomere stability in yeast. Here, we demonstrate several unexpected findings that underscore the complexity that surrounds hDna2 and support our proposal to study this protein in human cells. Specifically, we have shown that hDna2 localizes to the mitochondria and contributes to efficient mitochondrial DNA replication and repair (21). This work is in agreement with a previous report that claimed that hDna2 is restricted to the mitochondria (68). However, we also find that hDna2 1) localizes to the nucleus (21);2) is found within DNA damage foci;3) localizes to the telomere;4) interacts with the telomere binding protein TRF2;5) its depletion results in a hyper- recombination phenotype characterized by increased sister chromatid exchanges;and 6) its depletion leads to telomere shortening. We hypothesize that hDna2 plays a role in DNA replication within the nucleus where it is important for high fidelity DNA replication, DNA repair and telomere stability. Experiments proposed herein will delineate the biochemical activities of hDna2 that ensure high fidelity DNA replication and that impact telomere stability by determining whether these activities are required for faithful telomeric replication and/or capping. PUBLIC HEALTH RELEVANCE: Chromosomes within a normal human cell are under constant attack from agents that induce DNA damage. In addition, damage can also arise from the normal DNA replication process where the cellular DNA is copied. If left unrepaired, this DNA damage will lead to mutations and chromosome instability, which are defining characteristics of cancer cells. Because chromosomal integrity is of critical importance, numerous proteins have evolved to play complementary roles in surveying the DNA and correcting any mutations that arise. When these proteins undergo mutations that corrupt their activity, DNA damage persists and mutations accumulate. Given the importance of maintaining DNA integrity, many groups focus on defining mechanisms responsible for correcting DNA mutations and maintaining stable chromosomes. Maintenance of the chromosome ends (referred to as telomeres) is one factor that has a profound impact on chromosome stability. Indeed, loss of telomere stability leads to chromosomal fusion-breakage cycles that drive chromosome instability. Here, we study Dna2, a protein known to influence genomic stability in yeast. We show that hDna2 localizes to the "powerhouse" of the cell (i.e. the mitochondria) where it participates in DNA replication and repair. In addition, we show that hDna2 loss leads to chromosome instability and telomere shortening. Because telomere shortening has been linked to genomic instability, a hallmark of human cancer cells, understanding hDna2's role in DNA and telomere stability is paramount. The work proposed in this application will allow us to define how hDna2 contributes to telomere stability.